Conventionally, the measurement of the amount of an analyte in a sample using a redox reaction has been utilized for a wide range of applications. For example, such measurement has been utilized for measuring glycated proteins in applications such as biochemical analyses, clinical tests, and the like.
For instance, glycated proteins in blood, particularly glycated hemoglobin (HbA1c) in erythrocytes, are significant indicators in the diagnosis, therapy, and the like of diabetes, because they reflect the patient's past history of blood glucose levels. Glycated proteins in erythrocytes are measured using a redox reaction, for example, in the following manner.
First, erythrocytes are hemolyzed to prepare a sample. This hemolyzed sample is treated with a suitable protease or the like, and then treated with a fructosyl amino acid oxidase (hereinafter referred to as FAOD) so as to form hydrogen peroxide. The amount of the hydrogen peroxide formed corresponds to the amount of a glycated protein in erythrocytes. Then, a peroxidase (hereinafter referred to as POD) and a reducing agent further are added to the reaction solution, so that a redox reaction occurs between the hydrogen peroxide and the reducing agent with the POD as a catalyst. At this time, when a reducing agent that develops color when it is oxidized is used, the amount of the hydrogen peroxide can be determined by measuring the color developed. As a result, the amount of the glycated protein in erythrocytes can be determined.
However, various types of reducing substances, such as ascorbic acid (AsA) and bilirubin, usually are present in blood. Moreover, various types of reducing substances such as glutathione (GSH) are present in erythrocytes. These reducing substances may reduce the hydrogen peroxide, may inhibit the redox reaction, or may reduce the reducing agent after it develops color, so as to degrade the color. Therefore, there has been a problem that it is difficult to determine the amount of the glycated protein in erythrocytes accurately.
Also, there has been another problem that the accuracy of the measurement may deteriorate because the concentrations of the reducing substances contained in samples are not constant.
In order to avoid these problems, for example, various types of oxidizing agents have been added to samples. For example, JP 56(1981)-151358 A discloses a method of using halogen oxides, such as iodic acid or periodic acid, as oxidizing agents. JP 57(1982)-13357 A, JP 57(1982)-161650 A, JP 59(1984)-193354 A, JP 62(1987)-169053 A, and JP 3(1991)-30697 A also disclose methods of using complexes of metals such as cobalt, iron, cerium, etc. as oxidizing agents.
However, the influence of the reducing substances on the measurements cannot be avoided sufficiently even with the use of these oxidizing agents. In particular, these oxidizing agents performed poorly when the analyte is a component in erythrocytes.